1. Field of the Invention
The invention relates to substances which have the ability to bind to polynucleotides, e.g. DNA or RNA.
2. Discussion of the Background
Inhibition of the expression of single proteins by polynucleotides complementary to their mRNA's ("antisense mRNA's") is well documented (see, e.g., Izant and Weintraub, 1984; Rosenberg et al, 1985). This technique has even been employed for the differential deletion of the expression of a single protein within an operon (Pestka et al, 1984).
While this technique is best suited to in vitro studies, given the difficulties of delivering large oligonucleotides into intact cells and stabilizing them against metabolic degradation once they are introduced, Zamecnik and Stephenon have reported the use of an oligonucleotide (d(AATGGTAAAATGG)) complementary to the reiterated terminal sequences of Rous Sarcoma virus to inhibit the replication of the intact virus (Zamecnik and Stephenson, 1978, Stephenson and Zamecnik, 1978).
A number of laboratories have also investigated the feasibility of utilizing the polynucleotide probes to destroy their complementary target sequences by modifying the probes to contain attached alkylating agents (Vlassov et al, 1985; Norre et al., 1985a,b; Zarytova et al, 1986; Iverson and Dervan, 1987) or prosthetic groups that mediate the generation of diffusible oxygen radicals (Dreyer and Dervan, 1985; Chu and Orgel, 1985; Vlassov et al., 1985b; Boidot-Forge et al., 1986).
There are several problems with the use of such probe molecules for therapeutic intervention, or even for mechanistic studies. The change in free energy required for duplex formation argues that at least 12 nucleotides (and perhaps as many as 20) might be required for stable binding of a probe oligonucleotide to its target sequence (Naylor and Gilham, 1986; Mevarech et al., 1973, Noyes et al., 1979). Duplex formation is reversible and could also involve (transient) complex formation with sequences partially complementary to the probe (Wallace et al., 1979; Itakura and Riggs, 1980; Conner et al., 1983). This problem becomes more serious as the length of the probe is increased.
Additional problems also include the following. (1) Oligonucleotides are readily susceptible to degradation by nuclease activities present in all cells. (2) The delivery of unmodified oligonucleotides across cell membranes with reasonable efficiency is complicated by the negatively charged phosphate oxygen anions on the oligonucleotides. (3) The reactive groups employed thus far to permit a probe oligonucleotide to destroy their target sequences are such that they can also mediate destruction of the probes. (4) The chemical processes employed to date for destruction of target sequences are all limited to a single event (alkylating agents) or are unselective and probably unattainable under physiologic conditions.
In 1981, Letsinger and Schott described the preparation of a phenanthridinium TpT derivative (1) and demonstrated that it had an affinity for single-stranded poly A much greater than that of TpT itself. The mode of interaction of the modified dinucleotide with poly A was suggested to involve, in addition to Watson-Crick base pairing, both intercalation of the phenanthridine moiety and ionic interaction of one of the positively charged intercalator amino groups with the phosphate oxygen anion on poly A.
The work of Letsinger and Schott has been extended in the laboratory of C. Helene. First, these workers have demonstrated that the presence of an intercalator at the end of a probe oligonucleotide results in target sequence affinity at least as great as when the intercalator is tethered within the probe sequence.
Second, they demonstrated that the intercalator could be modified to mediate cleavage of the target strand, and would do so more efficiently under conditions where the probe was actually bound to the target (Doan, 1986). Unfortunately, the specific system developed was inefficient both for target binding and cleavage, and cannot be adapted to function under physiological conditions.
The third finding of this group is that oligonucleotide probes modified with intercalators can bind to mRNA's selectively in cell free systems and in Xenopus oocytes and thereby block translation of the derived proteins (Helene et al., 1985; Toulme et al., 1986; Cazenave et al., 1986.)
The issues of cell permeability and intracellular stability have been addressed by Miller and Ts'o. These workers have prepared oligonucleotides containing methylphosphonate linkages rather than the normal phosphodiester linkages in RNA and DNA (Miller et al., 1983a,b). Oligonucleotide analogs containing these linkages have been shown by Miller and Ts'o to ##STR1## be permeable to mammalian cells and to block mRNA production (Murakami et al., 1985, Blake et al., 1985a,b; Miller et al., 1985; Agris et al., 1986).
While the studies described by Miller and Ts'o are encouraging in it must be noted that this work also has limitations, which include:
the observation that the probe oligonucleotides could block mRNA translation only when they were complementary to the 5'-end or initiation codon regions of the mRNA (Blake et al., 1985a,b); PA1 the fact that the modified oligonucleotides are all complex mixtures of diastereomers due to the presence of chirality at the phosphonate P's (2.sup.n-1 isomers for an oligonucleotide n residues in length), each of which can be anticipated to have unique binding characteristics for the target RNA sequence (Pramanik and Kan, 1987).
Letsinger et al, "Nucleic Acids Research", vol. 14, no. 8, pp. 3487-3499 (1986), have described the contribution of certain lipophilic blocking group to polynucleotide affinity by probe molecules. This publication discloses several synthetic analogues of d-ApA containing a bulky lipophilic group (2,2,2-trichloroethoxy or 2,2,2-trichloro-1,1-dimethylethoxy), a small uncharged hydrogen-bonding group (amido), or a cationic phosphoramidate (2-aminoethylamido, protonated in neutral aqueous media).
Letsinger et al's study however fails to identify any relationship between the length/lipophilicity of the alkyl chain and the oligonucleotide binding properties of the probe, provides no teaching as to the molecular basis for the observed effects, does not identify ways to enhance/utilize the probe for biochemical intervention, and does not identify oligonucleotide N-alkylphosphoramidates as a discrete class of molecules with potentially useful properties.
There is therefore a need for a class of compounds which are capable of transport across cellular membrane, which are resistant to in vivo degradation, and which are capable of selectively binding polynucleotides (both DNA and RNA) both extracellularly and intracellularly. Such compounds would be useful in combating diseases by biochemical intervention at the DNA or RNA level.